Genetic Diversity and Genetic Heterogeneity of Bigfin Reef Squid “Sepioteuthis Lessoniana” Species Complex in Northwestern Pacific Ocean

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Genetic Diversity and Genetic Heterogeneity of Bigfin Reef Squid “Sepioteuthis lessoniana” Species Complex in Northwestern Pacific Ocean

Hideyuki Imai and Misuzu Aoki University of the Ryukyus Nara Women’s University Japan

1. Introduction The bigfin reef squid Sepioteuthis lessoniana Férussac, 1831 in Lesson (1830–1831) is widely distributed in the Indo-Pacific, where it is a very valuable fishery resource (Dunning, 1998). Thus, a lot of ecological research of this species were reported (e.g. Ikeda, 1933; Choe & Ohshima, 1961; Segawa, 1987; Ueta, 2003; Ikeda et al., 2009). Segawa et al. (1993a; 1993b) showed that within Sepioteuthis lessoninana have diferrences of egg chracteristics and reproductive trait in Ishigakijima Island. Izuka et al. (1994) reported an allozyme analysis found so-called S. lessoniana around Ishigakijima in Okinawa Prefecture, Japan, includes at least three biological species (Figure 1 & 2). Local fishers call the three species “aka-ika,” which has a red body, “shiro-ika” or “aori-ika,” which has a white body, and “kua-ika,” which is smaller than the other two. Of these, the range of “shiro-ika” extends to the coast of the main Japanese islands. This is the extent of its taxonomic classification thus far. This is due in part to the limited number of distinguishing morphological characters but also because the type specimens is no longer available and type locality has not been disignated (Lu et al., 1995; Jereb & Roper, 2006). This makes it difficult to determain whether genetically recognized species are undescraibed species or one of 13 known synonymies (Young, 2002). In this study, we treated “aka-ika” as Sepioteuthis sp. 1, “shiro-ika” as Sepioteuthis sp. 2, and “kua-ika” as Sepioteuthis sp. 3. A previous population genetics study found significant differences in the genetic heterogeneity of Sepioteuthis sp. 2 between Pacific Ocean and Japan Sea populations using allozyme analysis (Yokogawa & Ueta, 2000). Yokogawa and Ueta (2000) did not include the Okinawan Sepioteuthis sp. 2 population in their study. In addition, Pratoomchat et al. (2001) found no significant genetic heterogeneity between Japanese and Thai Sepioteuthis sp. 2 populations, while our present study tried significant differences in the genetic heterogeneity of the Japanese and Vietnumese Sepioteuthis sp. 2 populations. Recently, Aoki et al. (2008a) reported significant genetic heterogeneity between Japanese and Vietnumese populations of Sepioteuthis sp. 2 using DNA sequencing analysis of the mitochondrial noncoding region. Therefore, this study examined the genetic diversity (i.e., the average heterogeneity) and gene flow among Sepioteuthis sp. 2 populations using allozyme analysis and among

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152 Analysis of Genetic Variation in Animals populations of Sepioteuthis sp. 1 and Sepioteuthis sp. 3 using mitochondrial DNA noncoding region sequencing of populations from Japanese, Taiwanese, and Vietnamese waters.

A B C

D E F

Fig. 1. A: Sepioteuthis sp. 1, B: Sepioteuthis sp. 2, C: Sepioteuhis sp. 3, D: Sepioteuthis sp. 1 egg capsules with 5-13 (mean = 9) per capsule, E: Sepioteuthis sp. 2 egg capsules with 3-8 eggs (mode = 6) per capsule and F: Sepioteuthis sp. 3 egg capsules with consistently two eggs per capsule laid under dead table coral in shallow waters. Black bar indicated 50mm in length.

2. Materials & methods

2.1 Allozyme analysis of Sepioteuthis sp. 2 We collected 327 adults between September 1998 and June 2006 from Noto, Ishikawa, Japan (83 individuals), Mugi, Tokushima, Japan (51), the Goto Islands, Nagasaki, Japan (58), Nakagusuku, Okinawa (52), Keelung, Taiwan (23), and the Gulf of Tonkin, Vietnam (60). All

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Fig. 2. Electrophoretic patterns of asparate aminotransferase (AAT) of Sepioteuthis lessoniana complex. Lane 1-2: Sepiteuthis sp. 2, Lane 3-4: Sepioteuthis sp. 3 and Lane 5: Sepioteuthis sp. 1. These three species are clearly identified by the Aat-1* marker (Izuka et al. 1994). specimens were fresh and immediately sent to a refrigerator in the laboratory. The buccal bulb muscle was removed and kept frozen at –40°C until the allozyme analysis. Small pieces of liver and skeletal muscle were dissected from selected specimens and minced individually in an equal volume of distilled water on ice. Electrophoresis was conducted in a glass box with ice on top of it. The box was in a refrigerator at a constant voltage (250 V) until the Amido Black 10B marker moved seven cm from the origin. The allozymes were tested using 12.5% horizontal starch–gel electrophoresis and the two buffer systems described by Clayton and Tretiak (1972) and modified by Numachi (1989): citric acid N-(3-aminopropyl) diethanolamine (CAEA, pH 7) and citric acid N-(3-aminopropyl) morpholine (CAPM, pH 6). Each gel was sliced into six 1-mm-thick sheets with a wire gel cutter (Numachi, 1981) and stained for the enzymes aspartate aminotransferase (AAT), isocitrate dehydrogenase (IDHP), lactate dehydrogenase (LDH), phosphoglucomutase

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154 Analysis of Genetic Variation in Animals

(PGM), and phosphogluconate dehydrogenase (6PGD) according to Shaw and Prasad (1970), Numachi (1970a, b), and Taniguchi and Numachi (1978). The locus and gene nomenclature followed Shaklee et al. (1990). Polymorphisms involving several alleles with frequencies of more than 5% were tested at a significance level of 0.05 to determine whether they were consistent with Hardy–Weinberg equilibrium. The average heterozygosity H (Nei, 1978) was calculated as a measure of genetic diversity. The χ2 homogeneity test of allele frequency among samples was also performed.

2.2 Mitochondrial non-coding region of Sepioteuthis sp. 1 and sp. 3 In total, 116 Sepioteuthis sp. 1 were collected between April 2005 and September 2006 at three localities: Itoman, Okinawajima (49 individuals), Ishigakijima (38), and Keeling, Taiwan (29). An arm or part of the mantle muscles was kept in 90% ethanol and DNA was extracted with TNES 8M-Urea buffer. For Sepioteuthis sp. 3, 60 samples were collected between October 2005 and July 2006 from Nago, Okinawajima (30), and Ishigakijima (30). Crude DNA was extracted by TNES 8M Urea buffer and proteinase K digestion followed by a phenol- chloroform isoamyl method described Imai et al. (2004). We analyzed the noncoding region 2 (NC2) between the Ala and Trp transfer RNAs (tRNAs). The original primers SL-Ala (5'-GGTAACCCTTTCTGTATGATTGC-3') and SL-Trp (5'-AAAGACCTTGAAAGTCTTCAG-3'), which target a portion of tRNA-Ala and tRNA- Trp, respectively, were used with the polymerase chain reaction (PCR) to amplify NC2 (Aoki et al., 2008a). The PCR reactions were performed using BIOTAQ (Bioline, UK). A GeneAmp 9700 (Applied Biosystems, USA) thermal cycler was used with the following setting: 94°C for 120 s, followed by 30 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 45 s. The PCR products were purified using a PCR Product Pre-sequencing Kit (USB, USA). The nucleotide sequences were determined using ABI 3700 (Applied Biosystems, USA) genetic analyzers. All sequences were initially aligned using ClustalX ver. 1.83.1 (Thompson et al., 1997) and then edited manually using MacClade4 ver. 4.08 (Maddison and Maddison, 2005). The haplotype diversity h (Nei, 1987) and nucleotide diversity π (Tajima, 1983) within populations were calculated using Arlequin ver. 2.000 (Schneider et al., 2000). An analysis of molecular variance (AMOVA; Excoffier et al., 1992) was used to test the population structure within species for Sepioteuthis sp. 1 using Arlequin. For Sepioteuthis sp. 3, AMOVA could not be performed because there were fewer than three localities. Therefore, homogeneity was tested using the chi-square randomization method (Monte Carlo simulation) with 100,000 randomizations of the data (Roff and Benzen, 1989). Significance thresholds were Bonferroni-corrected for multiple pairwise comparisons. Relationships of haplotypes were assessed using a minimum spanning tree created via the Minspanet algorithm in Arlequin and drawn by hand.

3. Results and discussion 3.1 Allozyme analysis of Sepioteuthis sp. 2 Regarding the eight loci for the six enzymes analyzed, the five loci Aat-1*, Idhp-1*, Ldh-1*, Mdh-1*, and Mdh-3* showed no differences among and within localities, and no genetic

www.intechopen.com Genetic Diversity and Genetic Heterogeneity of Bigfin Reef Squid “Sepioteuthis lessoniana” Species Complex in Northwestern Pacific Ocean 155 polymorphism was recognized. Two Mdh-2* heterozygotes were found in Vietnam, although the frequency was 0.017; therefore, it was not considered a polymorphic allozyme locus (Table 1). Genetic polymorphism was detected within a locality for Pgm* and 6pgd*. The polymorphic allozyme loci were in Hardy–Weinberg equilibrium at the localities. Most of the alleles linked to a locus were monomorphic. A marked excess of homogeneity was found. The average observed heterozygosity H=0.005–0.052 was similar to the values of H=0.037 reported by Izuka et al. (1996) and 0.052–0.070 by Yokogawa and Ueta (2000). Other loliginid species have similar heterozygosity values: Loligo pealeii, H=0.006; Lolliguncula brevis, H=0; L. plei, H=0 (Garthwaite et al., 1989); Ommastrephes bartrami, H=0.004; Sthenoteuthis oualaniensis, H=0.011; Todarodes pacificus, H=0.043; Loliolus japonica, H=0.030 (Fujio & Kawada, 1989); L. vulgaris reynaudii, H=0.030; L. gahi, H=0.059 (Carvalho & Loney, 1989); L. bleekeri, H=0.003 (Suzuki et al., 1993); and L. chinensis, H=0.006–0.009 (Yeatman and Benzie, 1993). The family Loliginidae appears to be characterized by low genetic diversity.

Okinawajima

Table 1. Allele frequencies at eight loci and indices of genetic heterozygosities within six localities of Sepioteuthis sp. 2.

No allele frequency gap was observed among different localities for the polymorphic allozyme loci 6pgd* allele frequency. In contrast, a significant difference was detected between Pgm* in the Japanese and Vietnamese localities (Table 2). This result differed greatly from that of Pratoomchat et al. (2001), who found the same gene pool in Thailand and Nagasaki, while our result supported the result of Aoki et al. (2008). Pratoomchat et al. (2001) used Pgm* and 6pgd*, but the results might have been influenced by differences in the electrophoresis buffer. No difference in allele frequency was observed among localities in Japan. Yokogawa and Ueta (2000) showed replacement of Ldh-4* between the main island Japan Sea and Pacific sides, although the allozyme band pattern shown in that paper may have been manipulated, and we find the results suspect. Therefore, we examined Ldh-4* with a fresh sample following the advice of Dr. Yokogawa, and we did not find it. Pratoomchat et al. (2001) cited Yokogawa and Ueta (2000), but did not detect Ldh-4*. When Ldh-4* was eliminated, no difference existed between the Japan Sea and Pacific sides. Aoki et

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156 Analysis of Genetic Variation in Animals al. (2008a) could not show a difference between the Japan Sea and the main island Pacific side, even on analyzing the mitochondrial noncoding region sequence. If Ldh-4* of Yokogawa and Ueta (2000) is repeatable, the difference in a highly polymorphic marker among populations would not always be detected. For example, regarding Theragra chalcogramma, a restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNA and microsatellite DNA could not identify the difference among populations seen in the allele frequency of the superoxide dismutase (SOD) allozyme marker (Iwata, 1975; Mulligan et al., 1992; Bailey et al., 1999; Chow, 2001). Our finding of gene flow between Taiwanese and Japanese localities detected in the allozyme analysis of Sepioteuthis sp. 2 was not consistent with the sequencing analysis of the mitochondrial noncoding region by Aoki et al. (2008a), perhaps because of the low level of polymorphic loci for the allozyme analysis. In addition, noncoding regions such as the mitochondrial control region accumulate more variation than allozyme markers. The relative smallness of the effective population size (female) with nuclear DNA made it easier to detection interpopulation genetic differentiation by genetic drift (Williams et al., 2002). Therefore, Aoki et al. (2008a) used the mitochondrial noncoding region and found low genetic diversity in Japanese waters. Furthermore, Aoki et al. (2008a) revealed the independence of gene flow within the populations in Japanese waters from others.

Table 2. P-value in allele frequencies (Pgm*; above diagonal) and Nei’s genetic distance (below diagonal) among six localities of Sepioteuthis sp2. Bonferroni correction P<0.05.

3.2 Mitochondrial non-coding region of Sepioteuthis sp. 1 We sequenced 552 base pairs (bp) of the NC2 sequence for 116 Sepioteuthis sp. 2 specimens from three localities. From a total of 35 haplotypes, 23 variable sites were identified (Table 3). One haplotype was shared among three localities, and the remaining 31 haplotypes were each specific to a single locality. Among the populations, 23.3% of the samples belonged to haplotype no. 1, which was the major haplotype in all Japanese localities. In contrast, haplotype no. 3 was the major haplotype in Taiwan (Figure 3). The haplotype diversity (h) ranged from 0.7994 for Ishigakijima to 0.8665 for Okinawajima, and the nucleotide diversity (π) varied from 0.0035 for Ishigakijima to 0.0052 for Okinawajima (Table 4). Among the three Sepioteuthis sp. 1 localities, the level of genetic diversity did not differ much. The genetic diversity of Sepioteuthis sp. 1 was similar to that of Sepioteuthis sp. 2: h=0.8972, π=0.0124 in Taiwan and h=0.6828, π=0.0077 in Vietnam. The Japanese values of h=0.2583, π=0.0024 indicate that Japan has three times more genetic diversity than haplotype diversity.

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Table 3. Haplotype distribution and variable sites of mitochondrial NC2 region of Sepioteuthis sp. 1 among three localities.

Table 4. Haplotype diversity and nucleotide diversity of Sepioteuthis sp. 1 among three localities.

The AMOVA indicated that the genetic variation over all of the Japanese localities was 34.87%, whereas the within-locality variation was 65.13% (p<0.01 [Table 5]). The estimated pairwise Fst values for the three pairs of three localities ranged from 0.0538 to 0.5329. All combinations of locality samples had significant pairwise Fst values (p<0.05 [Table 6]). Therefore, each locality had an independent population with restricted gene flow, concurring with Aoki et al. (2008a). Sepioteuthis sp. 2 had gene flow within the territorial waters of Japan and showed genetic homogeneity. The relationships among the haplotypes

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158 Analysis of Genetic Variation in Animals

Fig. 3. Pie chart representation of the haplotype frequencies of Sepioteuthis sp. 1 of the three localities.

Table 5. Analysis of Molecular Variance on pairwaise differences and P-value of Sepioteuthis sp. 1 among three localities is the probability of a more extreme variance component.

Table 6. Pairwise Fst and associated probability (P) of Sepioteuthis sp. 1 among three localities. Fst values are below the diagonal and corresponding P values are above the diagonal. Bonfferroni correction P<0.05.

www.intechopen.com Genetic Diversity and Genetic Heterogeneity of Bigfin Reef Squid “Sepioteuthis lessoniana” Species Complex in Northwestern Pacific Ocean 159 were represented on a minimum spanning tree, and the shape indicated that the population had long-term stability (Figure 4).

Fig. 4. Minimum spanning tree among 35 hapolotypes of Sepioteuthis sp. 1.

3.3 Mitochondrial non-coding region of Sepioteuthis sp. 3 We sequenced 557 bp NC2 sequences for 60 Sepioteuthis sp. 3 specimens from two localities. From a total of 15 haplotypes, 13 variable sites were identified (Table 7). Seven haplotypes were shared between the two localities, and the remaining eight were specific to a single locality. Overall, 31.6% of the samples belonged to haplotype no. 1, which was not the major haplotype in both localities. Haplotype no. 2 was the major haplotype in Okinawajima (Figure 5). The haplotype diversity (h) ranged from 0.7103 for Ishigakijima to 0.8828 for Okinawajima, and the nucleotide diversity (π) varied from 0.0037 for Ishigakijima to 0.0044 for Okinawajima (Table 8). The level of genetic diversity was similar to that of Sepioteuthis sp. 1. Significant heterogeneity was observed between the Okinawajima and Ishigakijima populations (χ2=23.89, p<0.01). Therefore, these two populations could be distinguished by the haplotype frequency. Izuka et al. (1996) reported that each population was genetically independent based on the allozyme analysis for Ishigakijima and the Ogasawara Islands. These results suggest that Sepioteuthis sp. 3 does not experience larval dispersal, but completes its life history within coral reefs. The relationships among the haplotypes were represented on a minimum spanning tree, and the shape indicated that haplotypes could not be divided into clusters (Figure 6).

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160 Analysis of Genetic Variation in Animals

Table 7. Haplotype distribution and variable sites of mitochondrial NC2 region of Sepioteuthis sp. 3 between Okinawajima and Ishigakijima Island.

Table 8. Haplotype diversity and nucleotide diversity of Sepioteuthis sp. 3 between Okinawajima and Ishigakijima Island.

4. General discussion In this research showed that genetic differentiation between Okinawajima and Ishigakijima population was identified for Sepioteuthis sp. 1 and Sepioteuthis sp. 3. The result showed that Sepioteuthis sp. 1 and sp. 3 prefer coast lines as habitat that limit periodic dispersal of larva and adult among islands. The result showed that there is no gene flow of Sepioteuthis sp1 between Ishigakijima and Taiwan, as Aoki et al. (2008a) showed for Sepioteuthis sp. 2. Geographical distance of these two areas is 300 km, which has no difference of the geographical distance between Okinawajima and Ishigakijima Island. However, genetic structure differentiation between Ishigakijima and Taiwan is bigger than that of Okinawajima and Ishigakijima. Thus, gene flow between Ishigakijima and Taiwan was disturbed for long period. Kuroshio Current possible is possibly disturbing the gene flow Kuroshio Current is warm current with the surface speed of 2m per second, strong flow that moves more than 50 million tons of water. The current axis starts from north equatorial countercurrent, go up towards north between Taiwan and Yonagunijima, through Tokara strait and flows into southern coast of main island Japan (Figure 7). The width between Taiwan and Yonagunijima is small. Kuroshio Current go up to the north along with

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Fig. 5. Pie chart representation of the haplotype frequencies of Sepioteuthis sp. 3 of the two localities. continental shelf. The current split around Kyushu to Tsushima Current along with Japan Sea and main current that goes along with Pacific coast of main island, Japan. The current disperse when it goes to north, the flow of Kuroshio Currnet may take them to very northern part, that has lower temperature of ocean water. Geographical cal distribution of Panulirus longipes is another example in which Kuroshio Current is a barrier current between Taiwan and Ryukyu islands (Sekiguchi & Inoue, 2010). Accordingly, several marine organisms, Uca arcuata and Siganus guttatus shows different genetic structure between Ryukyu Archipelago and Taiwanese population showing no gene flow (Aoki et al., 2008b; Iwamoto et al., 2012). These genetic sturucture pattern may suggest the influence of Kuroshio Current. Especially squids has short longevity, some of the species has only one spawning season in a life. Drastic environmental change and some other accidental events may destroy population. In order to raise the fitness of squids, water temperature and appropriate environment of growth phase are essential (O’Dor & Coelho, 1993). Kuroshio Current may supply appropriate temperature and abundant feed resources for Sepioteuthis spp habitat. The comparative study of Sepioteuthis sp. 2 reported by Aoki et al. (2008a) and Sepioteuthis sp. 1 MST shape shows that these two species can belong to each clade of Japan and Taiwan.

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162 Analysis of Genetic Variation in Animals

Fig. 6. Minimum spanning tree among 15 hapolotypes of Sepioteuthis sp. 3.

120º 130º 140º Russia 45º

Japan Sea

Ishikawa Pref.

Korea Japan

Tsushima strait

China Tokushima Pref. 30º Nagasaki Pref. Tokara strait Kuroshio Current Pacific Ocean

Okinawajima Taiwan Ishigakijima

Fig. 7. The location of the main pathway of Kuroshio Current and sampling sites.

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5. Conclusion We analyzed the genetic heterogeneity of populations of Sepioteuthis sp. 1 and Sepioteuthis sp. 3 in Okinawan waters using DNA analysis. The genetic diversity of Sepioteuthis sp. 1 and Sepioteuthis sp. 3 was higher than that of Sepioteuthis sp. 2 from Japanese waters. Moreover, the genetic heterogeneity of the populations differed significantly. The difference in genetic heterogeneity means that Sepioteuthis sp. 1 and Sepioteuthis sp. 3 do not have a large gene pool. We postulate that the reason for the genetic differentiation is that these two species prefer coastal habitats. Our results indicate that the Japanese populations of Sepioteuthis sp. 2 have very low genetic diversity compared to those of Taiwan and Vietnam. The minimum spanning tree showed that the Japanese populations were of the radiation type, implying that the Japanese populations had experienced founder effects. The genetic heterogeneity in the Japanese populations was slightly different using AMOVA. This suggests that the mitochondrial noncoding region of the Japanese population lacks sufficient genetic diversity to assess the genetic heterogeneity in the Japanese populations. Moreover, our results suggest that only limited gene flow has occurred between the Ishigakijima and Taiwan populations of Sepioteuthis sp. 1 and Sepioteuthis sp. 2, implying the presence of barriers to gene flow. Kuroshio Current, a prominent current in this area, which moves at a rate of nearly 50 million m3/s, may prevent dispersal from Taiwan to Ishigakijima. Lastly, it should be noted that Prof. Segawa’s contribute to ecological research for Sepioteuthis lessoniana complex. Further study should focus on resolute species complex as soon as possible to develop ecological research of Sepioteuthis spp. It is necessary to identify species identification marker, however, frequently used allozyme marker needs fresh samples. Finding DNA marker that can be used for ethanol sample will be useful. DNA marker development should use allozyme marker of Izuka et al. (1994) and Triantafillos and Adams (2005) as the standard specimens. Allozyme analysis is the method to detect nuclear DNA polymorphism by the detection of enzyme molecule polymorphism. It cannot show nucleotide base-substitution mutation when it does not have amino-acid sequence variation. The different condition of electrophoresis buffer may produce different results, even though it is worth noting that it is a reliable tool to find cryptic species (Imai, 2006). The first author of this paper, Imai, H is currently working on development of species identification using DNA marker with some other researchers.

6. Acknowledgment We thank Prof. Y. Ikeda of the Faculty of Science, University of the Ryukyus, Dr. Y. Ueta of the Fisheries Research Institute, Tokushima Agriculture, Forestry, and Fisheries Technology Support Centre; Prof. T. Y. Chan and Dr. M. Mitsuhashi of the Institute of Marine Biology, National Taiwan Ocean University; Dr. B. K. K. Chan of the Research Centre for Biodiversity, Academia Sinica; Mr. T. Higa of the Nago Fishery Cooperative Association; Mr. Y. Yonamine of the Yaeyama Fishery Cooperative Association; and Mr. S. Nagata of Ryukyu-Taiyo Inc. for sample collection. We also thank Dr. K. Yokogawa Kagawa Prefecture residing for kindly advice of Ldh-4 detection and Ms. K. Hirouchi for checking English the manuscript.

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164 Analysis of Genetic Variation in Animals

7. References Aoki, M.; Imai, H. & Ikeda, Y. (2008a). Low genetic diversity of oval squid, Sepioteuthis cf. lessoniana (Cephalopoda: Loliginidae), in Japanese waters inferred from a mitochondrial DNA non-coding region. Pacific Science, Vol. 62, pp. 403-411. Aoki, M.; Naruse, T.; Cheng, J; Suzuki, Y. & Imai, H. (2008b). Low genetic variability in an endangered population of fiddler crab Uca arcuata on Okinawajima Island: analysis of mitochondrial DNA. Fisheries Science, Vol. 74, pp. 330-340. Bailey, K.; Quinn, M.; Benzie, P. & Grant, W. S. (1999). Population structure and dynamics of walleye pollock, Theragra chalcogramma. Advance in Marine Biology, Vol. 38, pp. 177-255. Carvalho, G. R. & Loney, K. H. (1989). Biochemical genetic studies on the Patagonian squid Loligo gahi d’Orbrigny. 1. Electrophoretic survey genetic variability. Journal of Experimental Marine Biology and Ecology, Vol. 126, pp. 231-241. Choe, S. & Ohshima, Y. (1961). On the embryonal development and growth of the squid, Sepioteuthis lessoniana Lesson. Venus ( Japanese Journal of Malaclogy), Vol. 21, pp. 462-476. Chow, S. (2001). Stock identification, in Tanaka, S, et al. (Ed.), Textbook for stock analysis method, 18-27, Japan Fisheries Resource Conservation Association, Tokyo [in Japanese]. Clayton, J.W. & Tretiak, D.N. (1972). Amine-citrate buffers for pH control in starch gel electrophoresis. Journal of the Fisheries Research Board of Canada, Vol. 29, pp.1169-1172. Excoffier, L.; Smouse, P. E. & Quattro, J. M. (1992). Analysis of molecular variance inferred from metric distance among DNA haplotypes: Application to human mitochondrial DNA restriction data. Genetics, Vol. 131, pp. 479–491. Dunning, M. C. (1998). Loliginidae, in Carpenter, K. E. & Niem, V. H. (Ed.), The living marine resources of the western central Pacific, 764-780, FAO species identification guide for fishery purposes, Food and Agriculture Organization of the United Nations, Rome. Fujio, Y. & Kawada, G. (1989). Genetic differentiation and variability in squids, in Report on the genetic assessment project (Ed.), Population analysis of marine organisms by isozyme analysis, 508–523, Japan Fisheries Resource Conservation Association, Tokyo [in Japanese]. Garthwaite, R. L.; Berg Jr., C. J. & Harrigan, J. (1989). Population genetics of the common squid Loligo pealei LeSueur, 1821 from Cape Cod to Cape Hatteras. Biological Bulletin, Vol. 177, pp. 287-294. Ikeda, H. (1933). Sex-correlated marking in Sepioteuthis lessoniana Férussac. Venus( Japanese Journal of Malaclogy), Vol. 3, pp. 324-329. Ikeda, Y.; Imai, H.; Sugimoto, C. & Oshima, Y. (2009). Egg case containing three ova of oval squid Sepioteuthis lessoniana in Okinawa Island of the Ryukyu Archipelago. Aquaculture Science, Vol. 57, pp. 631-634. Imai, H. (2006). Animal diversity of bay and tidal in Ryukyu Archipelago, in Biodiversity of coral reef and island ecosystems of the Ryuykus, 21st Centry COE Program, University ot the Ryukyu (Ed.), 35-47,. Tokai University Press, Tokyo [in Japanese]. Imai, H.; Cheng, J. H.; Hamasaki, K. & Numachi, K. (2004). Identification of mud crabs (genus Scylla) using ITS-1 and 16S rDNA markers. Aquatic Living Resources, Vol. 17, pp. 31–34. Iwamoto, K.; Chang, C.; Takemura, A. & Imai, H. (2012). Genetically structured population and demographic history of the goldlined spinefoot Siganus guttatus in northwastern Pacific. Fisheries Science, Vol. 78, in press [doi: 10.1007/s 12562-011- 0455-3].

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Iwata, M. (1975). Genetic identification of walleye pollock (Theragra chalcogramma) populations on the basis of tetrazolium oxidase polymorphism. Comparative Biochemical Physiology, Vol. 50B, pp. 197-201. Izuka, T.; Segawa, S. & Okutani, T. (1996). Biochemical study of the population heterogeneity and distribution of the oval squid Sepioteuthis lessoniana complex in southwestern Japan. American Malacological Bulletin, Vol. 12, pp. 129-135. Izuka, T.; Segawa, S.; Okutani, T. & Numachi, K. (1994). Evidence on the existence of three species in the oval squid Sepioteuthis lessoniana complex in southwestern Japan. Venus:( Japanese Journal of Malaclogy), Vol. 53, pp. 217-228. Jereb, P. & Roper, C. F. E. (2006). Cephalopods of the Indian Ocean. A review. Part I. Inshore squids (Loliginidae) collected during the International Indian Ocean Expedition. Proceedings ot the Biological Society of Washington, Vol. 119, pp. 91-136. Lu, C. C.; Boucher-Rodoni, R. & Tillier, A. (1995). Catalogue of types of recent Cephalopoda in the Muséum National d’Histoire Naturelle (France). Bulletin du Muséum national d’Histoire naturelle, Paris, 4e sétie, Vol. 17, pp. 307-343. Maddison, D. R. & Maddison, W. P. (2005). MacClade: A computer program for phylogenetic analysis. 4.08 OSX. Sinauer Associates, Inc. Sunderland, Massachusetts. Mulligan, T. J.; Chapman, R. W. & Brown, B. L. (1992). Mitochondrial DNA analysis of walleye polluck, Theragra chalcogramma, from the eastern Bering Sea and Shelikof Strait, Gulf of Alaska. Canadian Journal of Fisheries and Aquatic Sciences, Vol. 49, pp. 319-326. Nei, M. (1978). Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics, Vol. 89, pp. 583–590. Nei, M. (1987). Molecular evolutionary genetics, Columbia University Press, New York. Numachi, K. (1970a). Lactate and malate dehydrogenase isozyme pattern in fish and marine mammals. Bulletin of the Japanese Society of Scientific Fisheries, Vol. 36, pp. 1067-1077. Numachi, K. (1970b). Polymorphism of malate dehydrogenase and genetic structure of juvenile population in saury Cololabis saira. Bulletin of the Japanese Society of Scientific Fisheries, Vol. 36, pp. 1235-1241. Numachi, K. (1981). Simple method for preservation and scanning of starch gels. Biochemical Genetics, Vol. 19, pp. 233-236. Numachi, K. (1989). Identification of population of marne organisums by isozyme analysis, in Report on the genetic assessment project (Ed.), Population analysis of fish and shellfish by isozyme analysis, 42–63, Japan Fisheries Resource Conservation Association, Tokyo [in Japanese]. O’Dor, R. K. & Coelho, M. L. (1993). Big squid, big currents and big fisheries, in Okutani, T.; O’Dor, R. K. & Kubodera, T. (Ed.), Recent Advance in Fisheries Biology, 531-535, Tokai University Press, Tokyo. Pratoomchat, B.; Natsukari, Y.; Maki, I. & Chalermwat, K. (2001). Allozyme determination of genetic diversity in Japanese and Thai populations of oval squid (Sepioteuthis lessoniana Lesson, 1930). Lamar (Tokyo), Vol. 39, pp. 133-139. Roff, D. A. & Benzen, P. (1989). The statistical analysis of mitochondrial DNA polymorphisms: X2 and the problem of small samples. Molecular Biology and Evolution, Vol. 6, pp. 539-545.

www.intechopen.com

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Segawa, S. (1987). Life history of the oval squid, Sepioteuthis lessoniana in Kominato and adjacent waters, central Honshu, Japan. Journal of Tokyo University of Fisheries, Vol. 74, pp. 67-47. Segawa, S.; Hirayama, S. & Okutani, T. (1993a). Is Sepioteuthis lessoniana in Okinawa a single species?, in Okutani, T.; O’Dor, R. K. & Kubodera, T. (Ed.), Recent Advance in Fisheries Biology, 531-535, Tokai University Press, Tokyo. Segawa, S.; Izuka, T.; Tamashiro, T. & Okutani, T. (1993b). A note on mating and egg deposition by Sepioteuthis lessoniana in Ishigaki Island, Okinawa, southwestern Japan. Venus (Japanese Journal of Maralacology), Vol. 52, pp. 101-108. Sekiguchi, H. & Inoue, N. (2010). Laval recruitment and fisheries of the spiny lobster Panulirus japonicus couplong with the Kuroshio subgyre circulation in the western North Pacific: A review. Journal of Marine Bilogical Association of India, Vol. 52, pp. 195-207. Shaklee, J. B.; Allendorf, F. W.; Morizot, D. C. & Whitt, G. S. (1990). Gene nomenclature for protein-coding loci in fish. Transactions American Fisheries Society, Vol. 119, pp. 2–15. Shaw, C. R. & Prasad, R. (1970). Starch gel electrophoresis of enzyme- A compilation of recipes. Biochemical Genetics, Vol. 4, pp. 297-320. Suzuki, H.; Ichikawa, M. & Matsumoto, G. (1993). Genetic approach for elucidation of squid family, in Okutani, T.; O’Dor, R. K. & Kubodera, T. (Ed.), Recent Advance in Fisheries Biology, 531-535, Tokai University Press, Tokyo. Tajima, F. (1983). Evolutionary relationship of DNA sequences in finite populations. Genetics, Vol. 105, pp. 437–460. Taniguchi, N. & Numachi, K. (1978). Genetic variation of 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase, and glutamic-oxaloacetic transaminase in the liver of Japanese eel. Bulletin of the Japanese Society of Scientific Fisheries, Vol. 44, pp. 1351-1355. Thompson, J. D.; Gibson, T. J.; Plewniak, F.; Jeanmougin, F. & Higgins, D. G. (1997). The Clustal-X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Reseaech, Vol. 25, pp. 4876–4882. Triantafillos, L & Adams, M. (2005). Genetic evidence that the northern calamary, Sepioteuthis lessoniana, is a species complex in Australian waters. ICES Journal of Marine Science, Vol. 62, pp. 1665-1670 Ueta, Y. (2003). Ecology and stock management of oval squid, Sepioteuthis lessoniana. Japan Fisheries Resource Conservation Association, Tokyo [in Japanese]. Schneider, S.; Roessli, D. & Excoffier, L. (2000). Arlequin: A software for population genetics data analysis. Version 2.000. Genetics and Biometry Laboratory, University of Geneva, Switzerland. Williams S.T., Jara, J. A., Gomez, E. & Knowlton, N. ( 2002 ) The marine Indo-West Pacific break: contrasting the resolving power of mitochondrial and nuclear genes Integrative and Comparative Biology, Vol. 42 pp. 941-952 . Yokogawa, K. & Ueta, Y. (2000). Genetic analysis of oval squid (Sepioteuthis lessoniana) around Japan. Venus ( Japanese Journal of Malacology), Vol. 59, pp. 45-55. Young, R. E. (2002). Taxa associated with the family Loliginidae Lesueur, 1821. Version June 2002. http://tolweb.org/accessory/ Loliginidae_Taxa?acc_id=2326 in The Tree of Life Web Project, http://tolweb.org/. Yeatman, J. M. & Benzie, J. A. H. (1993). Crytic specification in Loligo from Northern Australia, in Okutani, T.; O’Dor, R. K. & Kubodera, T. (Ed.), Recent Advance in Fisheries Biology, 641-652, Tokai University Press, Tokyo.

www.intechopen.com Analysis of Genetic Variation in Animals Edited by Prof. Mahmut Caliskan

ISBN 978-953-51-0093-5 Hard cover, 360 pages Publisher InTech Published online 29, February, 2012 Published in print edition February, 2012

Analysis of Genetic Variation in Animals includes chapters revealing the magnitude of genetic variation existing in animal populations. The genetic diversity between and within populations displayed by molecular markers receive extensive interest due to the usefulness of this information in breeding and conservation programs. In this concept molecular markers give valuable information. The increasing availability of PCR-based molecular markers allows the detailed analyses and evaluation of genetic diversity in animals and also, the detection of genes influencing economically important traits. The purpose of the book is to provide a glimpse into the dynamic process of genetic variation in animals by presenting the thoughts of scientists who are engaged in the generation of new idea and techniques employed for the assessment of genetic diversity, often from very different perspectives. The book should prove useful to students, researchers, and experts in the area of conservation biology, genetic diversity, and molecular biology.

How to reference In order to correctly reference this scholarly work, feel free to copy and paste the following:

Hideyuki Imai and Misuzu Aoki (2012). Genetic Diversity and Genetic Heterogeneity of Bigfin Reef Squid “Sepioteuthis lessoniana” Species Complex in Northwestern Pacific Ocean, Analysis of Genetic Variation in Animals, Prof. Mahmut Caliskan (Ed.), ISBN: 978-953-51-0093-5, InTech, Available from: http://www.intechopen.com/books/analysis-of-genetic-variation-in-animals/genetic-diversity-and-genetic- heterogeneity-of-bigfin-reef-squid-sepioteuthis-lessoniana-species-com

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